Fluorescent semiconductor nanocrystals (quantum dots, QDs) combine several unique optical and spectroscopic properties that can be tuned via size and/or composition. See References 1-6. For instance, core-shell QDs, such as those made of ZnS-overcoated CdSe nanocrystals, exhibit narrow tunable emission throughout the visible spectrum, combined with high quantum yield and a remarkable resistance to chemical degradation. See References 7-12. These unique features have made them greatly appealing for use as in vivo and in vitro fluorescent probes in a variety of biomedical applications; these include cellular labelling, deep-tissue imaging, biochemical sensing and drug delivery vehicles. See References 11 and 13-27.
Highly fluorescent QDs with good control over size and crystallinity are mostly grown via reduction of organometallic precursors at high temperature and in the presence of hydrophobic coordinating ligands. See References 3, 4, and 7-10. This growth route yields nanocrystals that are only dispersible in organic solvents. A key requirement for a successful integration of these materials into biology is access to an effective and reproducible surface-modification strategy. See References 23 and 28-32. Cap exchange with bifunctional coordinating ligands has been used by several groups to promote the dispersion of various inorganic nanocrystals in buffer media. This strategy relies on the competitive removal of the hydrophobic capping molecules and their replacement with hydrophilic metal-coordinating ligands. See References 23. The strength of the ligand coordination onto the nanocrystal surface along with a strong affinity of the hydrophilic modules to buffer media ultimately control the long term colloidal stability of the QDs in biological environments.
Ligands presenting multiple thiol groups, such as derivates of dihydrolipoic acid (DHLA), greatly enhance the QD colloidal stability in various biological conditions, compared with those presenting mono-thiol or other weakly coordinating groups. See References 33-40. The multi-coordination interactions between the QD and multidentate ligands decrease the ligand dissociation rate from the nanocrystal surfaces, substantially improving the colloidal stability of the QDs in biological media. Nevertheless, thiol-terminated ligands tend to negatively affect the photoluminescence properties of the hydrophilic QDs. See Reference 41. Moreover, under ambient conditions (e.g., room temperature and light exposure) most thiol-based ligands can be affected by photo-oxidation during extended storage time, which cause ligand desorption from the QD surface. See References 28, 42, and 43. This problem becomes more serious at very low concentrations, since the dynamic equilibrium of coordination favors higher dissociation rates. To address some of these limitations, polymer ligands presenting multiple imidazole (or pyridine) groups have been developed as an alternative to thiol groups for coordination on the nanocrystal. See References 42 and 44-47. Imidazole is not affected by this oxidation problem and has been found to potentially enhance the QD emission. See Reference 48. However, imidazole and pyridine exhibit weaker coordination affinity to the nanocrystal surfaces than thiols. For instance, histidine-coated QDs can be easily exchanged by thiol-terminated ligands. See Reference 49. Furthermore, imidazole-based polymer ligands provide hydrophilic QDs that exhibit colloidal stability only in weakly acidic to alkaline pH since the imidazole groups tend to be protonated under acidic conditions (pH<6). See References 44 and 50. This limits their use for common and newer promising conjugation techniques (e.g., EDC coupling and hydrazide reaction are most efficient at pH 4-6). See Reference 51.
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